1. Field of the Invention
The present invention relates to an ultrafine inorganic phosphor, a specifically binding material labeled with this phosphor, and a detection method using this specific binding material
2. Description of the Related Art
In the fields of medicine and biology, there is a method in which a fluorescent substance consisting of organic molecules is used as a label, and fluorescence emitted by irradiation of ultraviolet radiation is observed with an optical microscope or a photodetector. This method has been conventionally used in the studies of viruses or reactions of enzymes or in clinical examinations. Of the methods of this type, a fluorescent antibody method is well known. This method uses an antibody to which an organic phosphor which emits fluorescence is bonded. The antigen-antibody reaction is compared to a keyhole and key relationship since the reaction has a very high selectivity. For this reason, the position of an antigen can be determined from the fluorescence intensity distribution.
In this field of art, a strong demand has recently arisen for studies of more precise antibody distributions by observing substances smaller than about 1 .mu.m. At present, successful realization of this depends upon the use of an electron microscope. In observations using electron microscopes, images are observed by using the difference in electron beam reflectances or transmittances among various positions of a specimen. Therefore, in observing an antibody with an electron microscope, elements with large atomic weights are utilized. For example, a molecule containing iron or osmium or a colloidal gold granule of about 1 to 100 nm is currently used as a label of the antibody. FIG. 1 schematically illustrates an antibody using a colloidal gold granule as a label, as an example of an antibody having a label. As in FIG. 1, a composite body of protein A 1 and a colloidal gold granule 2 bonds to an antibody 3. By an antigen-antibody reaction, this antibody 3 binds to a corresponding antigen 4. Therefore, by detecting the position of the colloidal gold granule 2 on a specimen, it is possible to determine the position of the antigen. It is also possible to observe two or more kinds of antigens at the same time by bonding two or more types of colloidal gold granules each having a different size to different kinds of antibodies. In this method, however, colloidal granules may overlap each other during the detection, and so a quantitative determination is difficult to make simply by measuring the number of the colloids.
Phosphors consisting of the above organic molecules include polystyrene spheres which have a particle size of several ten nanometers and emit red-, green-, and blue-lights, in addition to molecular organic fluorescent dyes. It has been conventionally known that the polystyrene spheres exhibit bright emissions upon being excited with ultraviolet radiation. Unfortunately, the emission ability of an organic phosphor, including this polystyrene sphere, is degraded upon irradiation of ultraviolet radiation or an electron beam, since the molecular bonds of the dye are readily destroyed. In fact, these organic phosphors cannot be put to use in observations of cathode-luminescence images because not only the original luminous efficacy is low but also the emission intensity is degraded too much to make observation repeatedly. Additionally, these organic phosphors lack storage stability, i.e., deteriorate during storage.
In contrast, inorganic phosphors are stable and deteriorate little upon irradiation of ultraviolet radiation and an electron beam. The particle sizes of inorganic phosphors currently being used in industrial applications are about 4 to 7 .mu.m for CRTs, about 3 to 8 .mu.m for fluorescent lamps, and about 3 to 9 .mu.m for X-ray purposes. The reasons why the particle size is set to these values are as follows. That is, most phosphors are used by being coated on a substrate, and the particle sizes as defined above facilitate this coating. A fluorescent screen is observed mainly by the human eye, and a satisfactory resolution can be obtained with particle sizes to this extent. In conventional manufacturing methods, the luminous efficacy can be readily optimized with particle sizes to this extent, while the luminous efficacy decreases with smaller particle sizes.
It is known that the luminous efficacy of an inorganic phosphor decreases if its particle size is decreased by milling or etching using an acid. The reason for this has been considered that the surface of each individual particle is covered with a non-light-emitting layer, and the volume ratio of this non-light-emitting layer to the entire particle increases if the particle size is decreased. In effect, FIG. 6 inserted in "Television Society Technical Reports ED-754", page 21 shows that the luminous efficacy is decreased to about 10 to 50% by decreasing the particle size from 7 .mu.m to about 1 .mu.m in phosphors manufactured by conventional manufacturing methods. When this is extrapolated, no emission can be expected with particle sizes of 100 nm or smaller.
When cathode-luminescence images are observed with an electron microscope, the images are blurred if the afterglow time of a phosphor is long. To obtain clear images, therefore, it is necessary to use a phosphor having as short an afterglow time as possible. It has been conventionally known that a phosphor doped with Eu as a luminescent center, e.g., Y.sub.2 O.sub.3 : Eu, has a high luminous efficacy and is easy to manufacture as a red phosphor. Unfortunately, this phosphor has a relatively long afterglow time up to milliseconds and is therefore unsuitable for observations of cathode-luminescence images.
As discussed above, in observing micro regions by using an electron microscope, it is difficult to perform quantitative measurements if a molecule or a colloidal gold granule containing elements having large atomic weights is used as a labeling agent. If a phosphor is used as the labeling agent instead of these substances, quantitative measurements are possible. However, organic phosphors deteriorate significantly upon irradiation with an electron beam or ultraviolet radiation or during storage. As inorganic phosphors, on the other hand, no phosphors having both a small particle size and a high luminous efficacy have been obtained yet.
Organic phosphors have been conventionally used in fields other than medicine, e.g., in the field of fluorescent inks under ultraviolet irradiation. However, these organic phosphors also have the problem that fluorescent intensity fades with use for long periods of time. For this reason, inorganic phosphors for fluorescent inks, which do not readily deteriorate have been desired. Unfortunately, the particle size of a phosphor for use in an ink is commonly 1 .mu.m or smaller. Therefore, as described above, no inorganic phosphor having both a small particle size and a high luminous efficacy has been obtained yet.